CN116622614A - Preparation method of fusarium graminearum spore liquid - Google Patents
Preparation method of fusarium graminearum spore liquid Download PDFInfo
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- 241000223195 Fusarium graminearum Species 0.000 title claims abstract description 60
- 239000007788 liquid Substances 0.000 title claims abstract description 43
- 238000002360 preparation method Methods 0.000 title claims abstract description 12
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 19
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- 238000007789 sealing Methods 0.000 claims description 16
- 230000001954 sterilising effect Effects 0.000 claims description 10
- 239000010413 mother solution Substances 0.000 claims description 9
- WIIZWVCIJKGZOK-RKDXNWHRSA-N chloramphenicol Chemical compound ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-N 0.000 claims description 8
- 229960005091 chloramphenicol Drugs 0.000 claims description 8
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Abstract
The application discloses a preparation method of fusarium graminearum spore liquid, and belongs to the technical field of plant protection. The method utilizes a corn straw culture medium or a wheat straw culture medium to promote the mass production of spores of fusarium graminearum, cultures for 5-7 days, the mycelia are red (wheat grain color of scab), a large amount of fusarium graminearum spore liquid can be obtained by filtering the mycelia, and after counting by a blood cell counting plate, the spore liquid concentration is regulated by using sterile water, so that fusarium graminearum spore liquid with proper concentration can be obtained. The application has the advantages of easily obtained raw materials, low cost, simple operation, universality and easy mass production, and can obtain a large amount of spores with uniform morphology.
Description
Technical Field
The application belongs to the technical field of plant protection, and particularly relates to a preparation method of fusarium graminearum spore liquid.
Background
Fusarium graminearum is a plant pathogenic fungus widely distributed in the world, and can infect wheat crops, rice, corn, sorghum, various weeds and the like. Fusarium graminearum is a main pathogenic bacteria causing wheat scab, the yield of diseased wheat is low, the quality is poor, toxins such as DON, NIV and the like can be produced after wheat is infected by the Fusarium graminearum, and poisoning phenomena such as dizziness, nausea, diarrhea and the like can be caused by eating infected wheat grains by people and livestock. Wheat scab is commonly spread in the middle and downstream Yangtze river of our country, mostly in high temperature, humid climates. Fusarium graminearum overwinters in plant residual species in the form of ascospores, becomes a primary infection source in next spring, forms and develops ascospores when the air temperature rises and the rainwater is abundant, and releases the ascospores, wherein the ascospores and the conidium are transmitted by wind, rainwater, irrigation water and the like, so that wheat scab burst in a large area at proper temperature and humidity, and serious threat is caused to agricultural production, food safety and human health. In researching the disease occurrence rule of wheat scab, the identification of wheat resistant varieties and the control technology of wheat scab, quantitative inoculation by using conidium is an indispensable means.
Fusarium graminearum grows in nutrition in various culture mediums, no conidium is produced, and the strain needs to be frequently transferred for long-term preservation, so that the strain variation is easy to cause, and the strain source is polluted. At present, the existing PDA culture medium and carnation culture medium basically do not generate conidium or have smaller spore yield, long period and easy variation.
Disclosure of Invention
Aiming at the problems in the prior art, the technical problem to be solved by the application is to provide a preparation method of fusarium graminearum spore liquid, which utilizes a corn straw culture medium or a wheat straw culture medium to promote the mass production of fusarium graminearum.
In order to solve the technical problems, the technical scheme adopted by the application is as follows:
a preparation method of fusarium graminearum spore liquid comprises the following steps: fusarium graminearum is cultivated by using a corn straw culture medium or a wheat straw culture medium, so that the Fusarium graminearum is promoted to produce a large number of spores.
Further, the preparation method of the wheat straw culture medium comprises the steps of cleaning, drying and cutting wheat straw into 3cm sections, weighing 50g, adding 1100mL of pure water into a 2000mL beaker, boiling until the liquid level is reduced to 1000mL, cooling, and filtering to fix the volume to obtain a wheat straw boiling liquid mother solution; diluting and subpackaging the mother solution according to a proportion, sealing by a sterile sealing film, carrying out heat-moisture sterilization at 121 ℃ for 30min, cooling, and adding chloramphenicol with the mass concentration of 1% to obtain the wheat straw culture medium.
Further, the preparation method of the corn stalk culture medium comprises the steps of cleaning corn stalk, drying, cutting into 3cm sections, weighing 50g, adding 1100mL of pure water into a 2000mL beaker, boiling until the liquid level drops to 1000mL, cooling, and filtering to fix the volume to obtain corn stalk boiling liquid mother liquor; diluting and subpackaging the mother solution according to a proportion, sealing by a sterile sealing film, carrying out heat-humidity sterilization at 121 ℃ for 30min, cooling, and adding chloramphenicol with the mass concentration of 1% to obtain the corn stalk culture medium.
Further, the mass concentration of the wheat straw culture medium and the corn straw culture medium is 1% -5%.
Further, the preparation method of the fusarium graminearum spore liquid comprises the following specific steps:
1) Strain activation: f, performing activation culture on fusarium graminearum on a PDA culture medium at 26-28 ℃ for 3-5 days;
2) Inoculating: when the colony grows to 3-5cm in diameter, punching at the edge by using a sterile puncher to obtain a plurality of bacterial cakes with the diameter of 3mm, picking and inoculating the bacterial cakes into prepared culture mediums by using a sterile inoculating needle, and inoculating 2 bacterial cakes in each culture medium;
3) Shake cultivation: placing the culture medium inoculated with the bacterial cake on a shaking table at 180rpm and keeping the temperature at 27 ℃ for light-shielding culture for 7 days;
4) Filtering hypha: filtering each culture medium with four layers of gauze to remove mycelium, centrifuging for 5min at 12000 r/min, removing supernatant, adding appropriate amount of sterile water, and mixing with vortex instrument to obtain Fusarium graminearum spore liquid.
Further, the PDA culture medium is prepared by boiling 200g peeled potato pieces in 1000mL boiling water for 30min, filtering with gauze, collecting potato juice, adding water to 1000mL, adding 20g glucose and agar powder, stirring, packaging, and sterilizing at 121deg.C for 30min.
The spore concentration obtained by the method is 36.25X10 5 Fusarium graminearum spore liquid of each mL.
Compared with the prior art, the application has the beneficial effects that:
(1) The application utilizes a wheat straw culture medium or a corn straw culture medium to promote the mass production of spores of fusarium graminearum, cultures for 5-7 days, the mycelia are red (wheat grain color of scab), a large amount of fusarium graminearum spore liquid can be obtained by filtering the mycelia, after counting by a blood cell counting plate, the concentration of the spore liquid is regulated by using sterile water, and the spore concentration is 36.25 multiplied by 10 5 Fusarium graminearum spore liquid of each mL.
(2) The operation method has the advantages of easily obtained raw materials, low cost, simple operation, universality and easy mass production, and can obtain a large number of spores with uniform morphology.
Drawings
FIG. 1 is a 100-fold magnification of a microscope of conidia after 7 days of cultivation of Fusarium graminearum in 5% corn straw medium;
FIG. 2 is a 400-fold magnification of the microscopic view of conidia after 7 days of culture of Fusarium graminearum in 5% wheat straw medium (A);
FIG. 3 is a 400-fold magnification of the microscopic view of conidia obtained after 7 days of cultivation of Fusarium graminearum in 5% corn straw medium (B);
FIG. 4 is a bar graph of spore yield of a series of straw liquid media.
Detailed Description
The present application will be further described with reference to specific embodiments for the purpose of making the objects, technical solutions and advantages of the present application more apparent. Unless otherwise indicated, all technical means used in the following examples are conventional means well known to those skilled in the art.
Fusarium graminearum strain Fusarium graminearum FG0609 to be tested is derived from the academy of agricultural sciences of Jiangsu province; the mung beans are green-removed mung beans purchased in the market; the straw is obtained after the fixation of healthy plants.
Test medium and preparation thereof:
1) 1%,2%, 5% mung bean broth medium: cleaning mung beans, drying surface moisture at 65 ℃, weighing 50g, adding 1100mL of pure water into a 2000mL beaker, boiling until the liquid level is reduced to 1000mL, cooling, and filtering to fix the volume to obtain mung bean boiling liquid mother liquor with the concentration of 5% (w/v). Diluting 5% mother solution into 1%, subpackaging 2% solution, sealing with sterile sealing film, sterilizing at 121deg.C for 30min under damp heat, cooling, and adding 1% chloramphenicol to obtain 1%,2% and 5% mung bean culture medium.
2) 1%,2%, 5% rice straw medium: cleaning and drying rice straw, cutting the rice straw into small sections of 3cm, weighing 50g, adding 1100mL of pure water into a 2000mL beaker, boiling until the liquid level is reduced to 1000mL, cooling, and filtering to fix the volume to obtain rice straw boiling liquid mother liquor with the concentration of 5% (w/v). Diluting 5% mother solution into 1%, subpackaging 2% solution, sealing with sterile sealing film, sterilizing at 121deg.C for 30min under damp heat, cooling, and adding 1% chloramphenicol to obtain 1%,2% and 5% rice culture medium.
3) 1%,2%, 5% wheat straw medium: cleaning, drying and shearing the wheat straw into small sections of 3cm, weighing 50g, adding 1100mL of pure water into a 2000mL beaker, boiling until the liquid level is reduced to 1000mL, cooling, and filtering to fix the volume to obtain the wheat straw boiling liquid mother liquor with the concentration of 5% (w/v). Diluting 5% mother solution into 1%, subpackaging 2% solution, sealing with sterile sealing film, sterilizing at 121deg.C for 30min under damp heat, cooling, and adding 1% chloramphenicol to obtain 1%,2% and 5% wheat culture medium.
4) 1%,2%, 5% corn stalk culture medium: cleaning corn straw, drying, cutting into small sections of 3cm, weighing 50g, adding 1100mL of pure water into a 2000mL beaker, boiling until the liquid level is reduced to 1000mL, cooling, filtering and fixing the volume to obtain the corn straw boiling liquid mother liquor with the concentration of 5% (w/v). Diluting 5% mother solution into 1%, subpackaging 2% solution, sealing with sterile sealing film, sterilizing at 121deg.C for 30min under damp heat, cooling, and adding 1% chloramphenicol to obtain 1%,2% and 5% corn culture medium.
5) The PDA culture medium preparation method comprises the following steps: cutting cleaned peeled potato into 200g, adding 1000mL of water, boiling in boiling water for 30min, filtering with gauze, collecting potato juice, adding water to 1000mL, adding 20g of glucose and agar powder, stirring, packaging, and sterilizing at 121deg.C under moist heat for 30min.
Example 1: spore production comparison test of fusarium graminearum on different liquid culture mediums
1. Activating: fusarium graminearum is activated and cultured on PDA culture medium at 26-28deg.C for 3-5 days.
2. Inoculating: when the colony grows to 3-5cm in diameter, a plurality of bacterial cakes with the diameter of 3mm are obtained by punching at the edge by a sterile puncher, and the bacterial cakes are picked and inoculated into the prepared liquid culture mediums by a sterile inoculating needle, and 2 bacterial cakes are inoculated into each culture medium.
3. Shake cultivation: the liquid medium inoculated with the bacterial cake was incubated for 7 days (27 ℃ C., 180 rpm) at constant temperature and in the dark on a shaking table.
4. Counting: filtering each liquid culture medium with four layers of gauze to remove hypha, centrifuging for 5min at 12000 r/min, discarding supernatant, adding appropriate amount of sterile water, mixing with vortex instrument, counting spores generated by the four liquid culture mediums with blood cell counting plate, and performing differential analysis on spore yield of Fusarium graminearum cultured on different liquid culture mediums.
5. As shown in Table 1, the yield of Fusarium graminearum in 5% corn stalk medium reached a maximum of about 36.25X10 5 The spore yield of each of the wheat straw culture medium with the concentration of 5% and the corn straw culture medium with the concentration of 2% is 17.5X10% 5 The culture medium has better spore production effect, the spore production amount of the rest culture medium is lower, and the spore production effect is not obvious. The wheat straw culture medium and the wheat straw culture medium have the advantages that the fusarium graminearum spore yield is obviously higher than that of other culture mediums, the spore yield is high, the effect is good, and compared with other culture mediums, the raw materials of the wheat straw culture medium and the wheat straw culture medium are easy to obtain, the cost is low, and the preparation method is simple.
TABLE 1 spore yield of Fusarium graminearum on different media (. Times.10) 5 personal/mL)
As shown in FIG. 1, the result is shown in a 100-fold enlarged view of the conidia under a microscope after the fusarium graminearum is cultured in 5% of corn culture medium for 7 days, the morphology is uniform, the quantity is large, and the corn culture medium has good induction effect on the conidia produced by the fusarium graminearum.
As shown in FIG. 2, the result is a 400-fold enlarged view (A) of the conidium under a microscope after the fusarium graminearum is cultured in 5% of wheat culture medium for 7 days, and the fusarium graminearum has uniform morphology and clear diaphragm, which indicates that the wheat culture medium has good induction effect on the conidium produced by the fusarium graminearum.
As shown in FIG. 3, the result is a 400-fold enlarged view (B) of the conidium under a microscope after the fusarium graminearum is cultured in 5% of the corn culture medium for 7 days, and the morphology is uniform and has a clear diaphragm, so that the corn culture medium has good induction effect on the conidium produced by the fusarium graminearum.
The results are shown in figure 2, which shows a column diagram of the spore yield of the series straw liquid culture medium, and show that 5% of the wheat culture medium and the corn culture medium have good effects of inducing fusarium graminearum to produce spores.
Comparative example 1
Taking mung bean culture medium as an example, compared with the culture medium selected by the application:
1. activating: fusarium graminearum is activated and cultured on PDA culture medium at 26-28deg.C for 3-5 days.
2. Inoculating: when the colony grows to 3-5cm in diameter, a plurality of bacterial cakes with the diameter of 3mm are obtained by punching at the edge by a sterile puncher, and the bacterial cakes are picked and inoculated into the prepared liquid culture mediums by a sterile inoculating needle, and 2 bacterial cakes are inoculated into each culture medium.
3. Shake cultivation: the liquid medium inoculated with the bacterial cake was incubated for 7 days (27 ℃ C., 180 rpm) at constant temperature and in the dark on a shaking table.
4. Counting: filtering each liquid culture medium with four layers of gauze to remove hypha, centrifuging for 5min at 12000 r/min, discarding supernatant, adding appropriate amount of sterile water, mixing with vortex instrument, counting spores generated by the four liquid culture mediums with blood cell counting plate, and performing differential analysis on spore yield of Fusarium graminearum cultured on different liquid culture mediums.
5. As shown in Table 2, fusarium graminearum in 1% and 5% of mung bean culture medium did not produce spores, and the spore production amounts of 2% of mung bean culture medium were 6.25X10 5 And each mL.
TABLE 2 Fusarium graminearum spore yield (. Times.10) on mung bean medium 5 personal/mL)
Comparative example 2
Effects of [ MB and CMC liquid media on Fusarium graminearum spore production levels, bi Yaqi et al, plant protection, 2020, 46 (6): 155-158] and the corresponding culture method, and the main processes and results are as follows:
1. fusarium graminearum is selected and placed on a new PDA culture medium, and purified and cultured for 8-10d at 25 ℃ until mycelia are fully distributed on the surface of the PDA culture medium.
2. A total of 3 carboxymethylcellulose (CMC) media was set up for the experiment as replicates.
3. Culturing for 7 days, wherein the average concentration of conidium is 0.54×10 5 And each mL.
Claims (7)
1. A preparation method of fusarium graminearum spore liquid is characterized in that fusarium graminearum is cultivated by a straw culture medium, and generation of a large number of spores of fusarium graminearum is promoted.
2. The method for preparing fusarium graminearum spore liquid of claim 1, wherein the straw culture medium comprises one or more of a wheat straw culture medium and a corn straw culture medium.
3. The method for preparing fusarium graminearum spore liquid according to claim 2, wherein the method for preparing the corn stalk culture medium comprises the following steps: cleaning corn straw, drying, cutting into small sections of 3cm, weighing 50g, adding 1100mL of pure water into a 2000mL beaker, boiling until the liquid level is reduced to 1000mL, cooling, filtering and fixing the volume to prepare corn straw boiling liquid mother liquor; diluting and subpackaging the mother solution according to a proportion, sealing by a sterile sealing film, carrying out heat-humidity sterilization at 121 ℃ for 30min, cooling, and adding chloramphenicol with the mass concentration of 1% to obtain the corn stalk culture medium.
4. The method for preparing fusarium graminearum spore liquid according to claim 2, wherein the method for preparing the wheat straw culture medium comprises the following steps: cleaning, drying and shearing wheat straw into small sections of 3cm, weighing 50g, adding 1100mL of pure water into a 2000mL beaker, boiling until the liquid level is reduced to 1000mL, cooling, and filtering to fix the volume to obtain a wheat straw boiling liquid mother liquor; diluting and subpackaging the mother solution according to a proportion, sealing by a sterile sealing film, carrying out heat-moisture sterilization at 121 ℃ for 30min, cooling, and adding chloramphenicol with the mass concentration of 1% to obtain the wheat straw culture medium.
5. The method for preparing fusarium graminearum spore liquid according to any one of claims 1 to 4, wherein the mass concentration of the corn straw culture medium or the wheat straw culture medium is 1 to 5 percent.
6. The method for preparing fusarium graminearum spore liquid according to any one of claims 1 to 4, comprising the specific steps of:
1) F, performing activation culture on fusarium graminearum on a PDA culture medium at 26-28 ℃ for 3-5 days;
2) When the colony grows to 3-5cm in diameter, punching at the edge by using a sterile puncher to obtain a plurality of bacterial cakes with the diameter of 3mm, picking and inoculating the bacterial cakes into prepared culture mediums by using a sterile inoculating needle, and inoculating 2 bacterial cakes in each culture medium;
3) Placing the culture medium inoculated with the bacterial cake on a shaking table at 180rpm and keeping the temperature at 27 ℃ for light-shielding culture for 7 days;
4) Filtering each culture medium with four layers of gauze to remove mycelium, centrifuging for 5min at 12000 r/min, removing supernatant, adding appropriate amount of sterile water, and mixing with vortex instrument to obtain Fusarium graminearum spore liquid.
7. The method of any one of claims 1-6, wherein the spore concentration is 36.25X10 5 Fusarium graminearum spore liquid of each mL.
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